The objective of this research is to advance the understanding of the molecular basis for regulation of gene expression of a metabolite regulated, hepatic enzyme using argininsuccinate synthetase (ASS) as a model system. Preliminary studies using the human cell line RPMI 2650 have demonstrated increased ASS activity when citrulline is substituted for arginine in the growth medium. We have isolated canavanine resistant (Canr) cell variants which have up to 200 fold increased ASS activity. Evidence for enzyme overproduction in the Canr cells includes increased ASS protein determined immunologically, increased newly synthesized ASS protein when intact cells are labeled with (35S)methionine and increased translatable mRNA for ASS. We have used mRNA enriched for ASS to isolate cDNA clones in pBR322. These clones will be tested using differential filter hybridization and hybridization-translation to identify cloned cDNA for ASS. Using nick-translation of cloned cDNA to make probes, the relative amounts of mRNA for ASS will be determined in wild-type cells growtn in citrulline, wild-type cells grown in arginine and in Canr cells using liquid hybridization and filter hybridization after gel electrophoresis of the RNA. The relative gene copy number for ASS will be determined in wild-type cells in Canr cells to determine if gene amplification is the basis for ASS overproduction. The gene copy number and restriction fragment pattern for ASS genomic DNA will be examined in various human tissues including liver. Cloned genomic DNA for ASS will be identified by screening of a DNA library with the cloned cDNA as a probe. DNA mediated gene transfer into cultured cells will be carried out using cloned genomic DNA and total cell DNA as appropriate. If gene amplification is the basis of ASS overproduction in Canr cells, we will focus on the mechanism of metabolite regulation of ASS. If gene amplification is not found, the mechanism of enzyme overproduction will be the main focus of the study.